Endothelial cells synthesize prostacyclin, one of the most potent antiplatelet agents. Arachidonic acid, the precursor for prostacyclin synthesis, is present as phospholipid in the cells. The availability of free arachidonic acid governs the synthesis of prostacyclin. The proposed research has three principal objectives relating to the synthesis of prostacyclin and prostaglandins in the endothelial cells: 1) investigation of the phospholipases activated (phospholipase A2 and phospholipase C), when the endothelial cells are stimulated to synthesize prostacyclin. We propse to examine the phospholipase activation by several structurally and functionally very different stimulators' bradykinin, thrombin, histamine and ionophore A23187. We also propose to study the influence of a variety of diversified physiological and non-physiological effectors on the activated phospholipases. These studies shall provide us with information on the possible regulatory mechanism of the various phospholipases; 2) characterization of the activated PI-hydrolyzing phospholipase A2, one of the very earlier enzymes activated; 3) elucidation of the mechanism of glucocorticoid inhibition of arachidonic acid release. Our approach involves double labeling the endothelial cells with [3H]arachidonic acid and the phospholipid precursors; [14C]stearic acid, inositol, choline or ethanolamine. Our perliminary experiments with this procedure have demonstrated that these approaches are feasible. We shall also employ a two-dimensional thin layer chromatographic technique developed in our laboratory to analyze the various phospholipids and their metabolites. This technique enables us to separate PC, PE, PI, PS, lysoPI, lysoPC, lysoPE, lysoPS, phosphatidic acid, 1,2-diacylglycerol and 1,3-diacylglycerol. Ultimately, the information obtained from these studies will yield a better understanding of the control mechanism of prostacyclin synthesis in endothelial cells. These studies are pertinent to an understanding of the contribution of arachidonic acid and its metabolites to normal hemostasis and to the pathophysiology of thrombosis and atherosclerosis and can provide a pharmacological basis for drug therapy toward these diseases.